ABSTRACT Our long-term goal is to understand steroid regulation of social interaction by studying limbic-hypothalamic neural circuits that control female sexually receptivity, lordosis. We demonstrated that within this circuit, estradiol membrane-initiated signaling activates an arcuate nucleus (ARH) to medial preoptic nucleus (MPN) projection responsible for transient ?-opioid receptor (MORs) activation needed for full sexual receptivity. These MOR neurons in turn project to the ventromedial nucleus of the hypothalamus (VMH) modulating lordosis behavior. The present proposal is based on these results and on results in male mice that show that the posterodorsal medial amygdala (MeApd) modulates social behavior. Because in females, MeApd projections affecting the social behavior, lordosis, are not well defined, we seek to characterize specific MeApd projections that modulate the ARH-MPN-VMH circuit underlying steroid activation of female sexual receptivity. We will test the general hypothesis that the MeApd gates estradiol-initiated opioid inhibition to modulate sexual receptivity. Three Specific Aims are proposed to functionally dissect this circuit: 1) Selective activation of intrinsic ARH NPY neurons, and MPN-projecting POMC terminals inhibit lordosis. This experiment formally tests that the sequential activation of NPY and POMC neurons inhibits lordosis. NPY-Cre and POMC-Cre neurons will be infected with a stimulatory Cre-dependent channelrhodopsin (ChR2) adeno-associated virus (AAV) and photostimulated in behaving female mice. 2) The MeApd differentially modulates sexual receptivity through glutamatergic projections to the MPN, and GABAergic to the VMH. We will use AAV-directed expression of ChR2, to stimulate, or halorhodopsin, to inhibit, glutamatergic and GABAergic MeApd outputs. Our expectation is that glutamatergic inputs to the MPN enhance the MOR-mediated inhibition of lordosis (asocial behavior), while GABAergic inputs to the VMH promote receptivity (social behavior), as measured by the lordosis response to stimulus males. 3) Sexual receptivity involves a switch from glutamatergic to GABAergic neuron activation in the MeApd. These experiments will determine if in females, as in males, the MeApd controls social behavior through shift from glutamatergic (asocial) to GABAergic (social) outputs. First, we will determine the pattern of glutamatergic and GABAergic neuron activation in the MeApd induced by estradiol, and estradiol + progesterone is associated with the change from nonreceptive/asocial behavior to receptive/social behavior. Finally, we will sequentially inhibit glutamatergic and GABAergic MeApd neurons in gonadally intact, cycling vGLUT-Cre and vGAT-Cre mice using a Cre-dependent halorhodopsin-AAV. Together, these experiments will provide a functional circuit analysis of how steroid signaling in the limbic-hypothalamic circuit leads a prototypic social interaction, lordosis behavior.